Reciprocal regulation between the ECM and associated epithelial cells is integral to development, homeostasis and disease. Somites are segmental precursors of the vertebral column and musculature that form via a mesenchymal to epithelial transition. Somite morphogenesis is dependent upon a Fibronectin ECM, the Fibronectin receptor Integrin ?5?1, the cell adhesion protein Cadherin 2 and bidirectional signaling via the receptor tyrosine kinase EphA4 and its membrane bound ligand Ephrin-B2a. These genes/pathways mediate cell-ECM adhesion, cell-cell adhesion and contact mediated cell repulsion, and our hypothesis is that the physical organizing activity of the somite boundary emerges via specific spatiotemporal intertwining of differential cell adhesion and ECM constrained cell repulsion. In Aim 1, fluorescence correlation spectroscopy (FCS) and fluorescence crosscorrelation spectroscopy (FCCS) will be used quantify protein diffusion and protein binding constants in vivo. These experiments will determine whether the segregation of these cell surface proteins occurs via diffusion and capture or active mobilization. Additionally, the roles of integrin ?5, cadherin 2 and ephrin-b2a in driving these changes in subcellular localization will be elucidated by performing FCCS in live mutant embryos. In Aim 2, a systems analysis of cell motion will be used to quantify tissue biomechanics during somite morphogenesis in wild-type and mutant embryos. In Aim 3, we quantify the relative levels of Integrin activation via cytoplasmic signals versus via positive feedback through the ECM. Positive and negative feedback between biological mechanisms creates network effects that are hard to predict a priori and difficult to fully explore experimentally. in silico modeling will be used to systematically examine the relationships between cell adhesion, cell-ECM adhesion and cell contact mediated repulsion in somite morphogenesis in order to help interpret and prioritize more resource intensive wet-lab experiments.